| Objective The study was designed to evaluate effect permeability of ulinastatin on the blood brain barrier (BBB) and Caveolin-1 (CAV-1)> Zonula Occludens-1 (ZO-1) expression on rats with 15 min of global cerebral ischemia/reperfusion (GCI/R) 6h,24h,48h, and to discuss the neuroprotective effect of Ulinastatin with the cerebral ischemia/ reperfusion injury.Methods The study was carried out on 108 Wistar rats, weights 240g-280g, which were randomly divided into three groups (n=12):sham group (S group), global cerebral ischemia/reperfusion model group (GCI/R group) and 10,000U/Kg ulinastatin intervention group (U group). In this study we used the Pulsinelli four-vessle occlusion (4-VO) model of global cerebral ischemia reperfusion to investigate the integrity of blood brain barrier with the Evans Blue (EB) staining at 15 min after ischemia and at 6h,24h,48h after reperfusion, respectively, to assess blood brain barrier permeability with MRI, and to observe the expression of and CAV-1ã€ZO-1 in hippocampus with immunohistochemical analysis and Western Blotting analysis.Results(1) We successfully generated the rat model of global cerebral ischemia reperfusion, in which rats lost consciousness during the period of 60s after the common carotid arteries being occluded, developed typical symptoms of cerebral ischemia including bilateral mydriasis, absence of corneal reflex, limb stiffness and loss of their righting reflexes.(2) EB staining showed that the integrity of BBB was damaged after cerebral ischemia reperfusion. The brain tissue of ischemia hemisphere was stained in blue, whereas no visible staining was found in the rat brain tissues from the sham group after cerebral ischemia-reperfusion 6h,24h, 48h, and quantitative measurements showed little EB content in the rat brain tissue of the sham group. The model group were found visible staining after ischemia-reperfusion 6h,24h,48h, and EB content significantly increased after reperfusion 6h, reached a peak at 24h, and then began to decline, as compared with the sham group (P<0.05). In Ulinastatin treatment group, the area of EB staining was significantly smaller, the exudation of EB decreased significantly after cerebral ischemia-reperfusion 6h,24h,48h, as compared with the model group at corresponding time points (P<0.05), but increased as compare with the sham group.(3) MRI showed the leakage of BBB after cerebral ischemia reperfusion injury. After cerebral ischemia-reperfusion 6h,24h,48h, there were no differences of MRI enhancement signal in Hippocampus CA-1 area, the lateral ventricle, the third ventricle and cortex area after injecting Gd-DTPA. The model group appeared significantly highlighted regions of Gd-DTPA after post-contrast at the corresponding areas and time points, as compared with the sham group (P<0.05). The highlighted regions of Gd-DTPA in Ulinastatin treatment group were significantly higher as compared with those of the sham group, but lower when compared with those of the model group (P<0.05).(4) The expression level of CAV-1 changed after cerebral ischemia-reperfusion injury. Immunochemical staining results showed that expression of CAV-1 was positively detected in rat Hippocampus region of the sham group at 6h,24h and 48h after cerebral ischemia-reperfusion. The positive staining of Caveolin-1 decreased significantly in the model group after reperfusion 6h,24h,48h, as compared with the sham group (P<0.05). In Ulinastatin group, the positive staining of CAV-1 was significantly less than those in the sham group, but was significantly stronger than those in the model group (P<0.05). The Western blotting results were consistent with observations by immunohistochemical anslysis. The expression of CAV-1 protein was significantly down-regulated in Hippocampus after reperfusion 6h,24h, and 48h, as compared with the sham group and the Ulinastatin group (P<0.05). In the Ulinastatin group, CAV-1 protein expression level was lower than that of the sham group.(5) The expression level of ZO-1 changed after cerebral ischemia-reperfusion injury. Expression of ZO-1 was positively detected in Hippocampus in the sham group after cerebral ischemia-reperfusion 6h, 24h,48h. The expression of ZO-1 was significantly less in the model group after reperfusion 6h,24h,48h, than those in the sham group (P<0.05), respectively. In the Ulinastatin group, expression of ZO-1 was significantly less than those in the sham group, but was significantly more than those in the model group (P<0.05). In addition, the expression of ZO-1 protein was significantly down-regulated in Hippocampus after reperfusion 6h,24h, and 48h, as compared with the sham group and the Ulinastatin group (P<0.05). In the Ulinastatin group, ZO-1 protein expression level was lower than that in the control group.Conclusion(1) The integrity of blood brain barrier after cerebral ischemia reperfusion injury was damaged, so that the permeability and leakage increased in the model group.(2) In this study, we observed such leakage in the following areas including Hippocampus. And we observed that the expression levels of CAV-1 and ZO-1 significantly reduced in the model group.(3) The expression levels of CAV-1 and ZO-1 in the Hippocampus, which are the components of blood brain barrier, were deceased after cerebral ischemia reperfusion. We believe that the reason for blood brain barrier damage might be related to the decrease of expression of CAV-1 and ZO-1 after global cerebral ischemia/reperfusion.(4) Our finding suggested that Ulinastatin could significantly improve the permeability of blood brain barrier after cerebral ischemia/reperfusion injury; and the underlying mechanism of action we believe was that Ulinastatin induced expressions of CAV-1 and ZO-1, thus reduced the permeability of the blood brain barrier, and prevented the cerebral blood capillary leakage. The long-term protective effect of Ulinastatin will be tested and observed in future study for longer period of time on rats with global cerebral ischemia reperfusion injury. |
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